Compromiso organizacional

The current concept of the mechanism of secretion envisages a complex process in which the breakdown of phosphatidylinositol (PI) in the plasma membrane plays a central role and leads to the generation of intracellular messengers [272, 302]. As stated above, receptor activation on the plasma membrane is coupled through a G- protein to polyphosphoinositide phosphodiesterase [223, 238]. This catalyses the hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP^) to the intracellular messengers: IP3, which mobilizes stored calcium and may promote the influx of external calcium,

perhaps working in conjunction with inositol-1,3,4,5-tetrakisphosphate [272, 303]; and DAG which stimulates PKC [302]. PIP; and phosphatidylinositol-4-phosphate (PIP) are formed as a result of PI phosphorylation by ATP-dependent specific kinases [304].

Rat mast cell granules have been shown to contain active PI kinase and PIP kinase (3051.

Using radiolabelled “ PO4 and ^H-inositol, the importance of the PI response was

first reported by Cockroft and Gomperts [306], and Kennerly et a! [307], independently in 1979. It was found that mast cell showed marked receptor-initiated enhancement of “ PO4 and ^H-inositol incorporation into PI and phosphatidic acid (PA), as well as

unexpectedly phosphatidylcholine (PC). Similar findings were also reported by Schellenberg in 1980 [308]. Based on the negative results in a study of ^H-glycerol incorporation, the author emphasized that the enhancement of incorporation of “ PO4 into

PI, PA, and PC by various non-cytotoxic releasers was not due to the de novo synthesis of these phospholipids.

The DAG production from PIP^ is normally transient and temporally corresponds to the formation of IP,, and it is often followed by a more sustained increase in the amount of DAG [302]. There is increasing evidence that the DAG formation from PC via the initial conversion of PC to PA by phospholipase D (PLD) is the major pathway of DAG production during mast cell activation [309-313].

The simultaneous increase in inositol phospholipid breakdown and histamine release by 48/80, Sub P, and mastoparan, has also been demonstrated [314-317].

Inositol triphosphate and calcium signalling

The second messenger IP,, which is released into the cytosol, has been shown to mobilize calcium via specific receptors from internal stores, such as endoplasmic reticulum (ER), and has also been suggested to induce influx of external calcium [268, 272, 318]. Using permeabilized rat peritoneal mast cells, it has been shown that the intracellular store of calcium is located in the ER, and IP, induces degranulation dose- dependently [268, 319].

The influx of external calcium may also require the presence of IP4 [272, 303].

There is growing evidence that inositol phosphates may have direct effects on calcium channels that allow influx of calcium through the plasma membrane. For example, the IPg-induced entry of calcium in lymphocytes may be mediated by a receptor which contains sialic acid and is localized in the plasma membrane [320]. In addition to the direct effects, IP3 may also induce calcium influx indirectly through a more complex

mechanism involving ER. Putney in 1986 [321] used the term "capacitative entry" to introduce the idea that the influx of external calcium seemed to be regulated by the calcium content of a portion of the ER lying close to the plasma membrane. It has

been shown in the mast cell that calcium entry is stimulated when the ER stores are artificially emptied by applying the calcium pump inhibitor thapsigargin or the calcium ionophore ionomycin [3181. When the ER is fully charged, the entry is prevented, but as soon as IP3 drains calcium out, the influx switches on automatically. This mechanism

is thought to account for the entry of calcium into mast cells [3181. A possible explanation for this capacitative mechanism is that IP3 receptor might function to

communicate information between the ER and plasma membrane [272]. The concept is that part of the IP3 receptor communicates with two calcium channels, one in the ER

and the other in the plasma membrane. The conformational state induced by IP3

binding opens the ER channel, and as the pool loses its calcium, the receptor may alter its conformation, leading to the opening of the plasma membrane calcium channel.

1.2-diacvlglvcerol and the activation of protein kinase C

PKC is widely distributed in the cytosol and membrane fractions of a variety of mammalian tissues, and there is evidence which suggests that PKC-mediated protein phosphorylations play a central role in stimulus-effect coupling in these tissues [302, 3221. The presence of PKC in RPMC and rat basophilic leukaemia cells has been reported, although the activity is much lower than that in the cerebral cortex or in the other peritoneal cells [323]. DAG, which is retained within the membrane environment, has been shown to activate PKC which in turn leads to phosphorylation of serine and threonine residues in a subset of proteins in various cell types, including mast cells [302, 305, 324-327]. Translocation of PKC into cell membrane is a cytosolic calcium concentration-dependent process that may be crucial for mast cell histamine release [326]. PKC, with its activation, has been proposed as a possible modulator for extracellular calcium-dependent stimuli as the result of the observations of enhanced calcium uptake and the potentiation of the response to antigen, A23187,and NaF [323- 325, 327] The enhanced calcium uptake also reflects that PKC may contribute to the replenishment of the intracellular calcium stores after the secretory response [325]. Moreover, it has been reported that histamine release induced by antigen (which is calcium-dependent), but not that by 48/80, is inhibited by pretreatment with PKC inhibitors, implicating the involvement of PKC in IgE-mediated release, but not in 48/80- induced release [328]. However, it has also been reported that the activation of PKC is not essential for mediator release induced by antigen stimulation in rat basophilic leukaemia 2H3 cells [329, 330]. Furthermore, findings from a study using permeabilized rat mast cells has suggested that the PKC activation is not required for exocytosis to

occur [3311. Thus the relationship between PKC activation and histamine release is not yet clear. It has been shown that there are more than one species of PKC molecule with multiple discrete subspecies ( 1 0 subspecies), and not all of them are calcium-

and/or DAG-dependent for their activation [3021.

The synthetic diacylglycerol, 1 -oleoyl-2-acetyl-glycerol (OAG) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) can interact at the same site as DAG and activate PKC [332, 3331. OAG or TPA (ie. activation of PKC) alone induces slow histamine release [325, 3341. Depending on the concentrations and incubation periods of OAG or TPA, synergistic and inhibitory effects of both compounds on histamine release induced by secretagogues have been reported [324, 325, 3351. Low concentrations and short incubations of OAG/TPA show potentiation in histamine release, whereas high concentrations and long incubations show inhibition. It has been shown that the inhibition of histamine release during PKC activation by phorbol esters is mediated through inhibition of PI breakdown and calcium mobilization, perhaps by phorbol esters [310, 3351. Alternately, it may be the true effects of PKC. Thus, the potentiation suggests that PKC is involved in the secretory process by the secretagogues, while the inhibition indicates a regulatory function [3251 (possibly desensitization). The slow histamine release induced by PKC activation may suggest that PKC may be important but not sufficient in mast cell activation.

1,2-Dioctanoyl-sn-glycerol (Dic8) is a synthetic 1,2-diglyceride that has been

reported to activate PKC [336, 3371.